Vaccines can slow the spread of the HIV/AIDS epidemic, and the identification of immunogens that induce neutralizing antibodies remains an important goal of HIV vaccine research. An increasing number of broadly neutralizing human monoclonal antibodies (mAbs) have been isolated from infected individuals who show remarkably broad and potent neutralizing responses. Coupled with past studies, these results support the idea that the human immune system can generate rare but potent protective antibodies to the virus. Much work, including our own, has focused on identifying forms of the HIV-1 envelope glycoprotein (Env) that bind to broadly neutralizing antibodies. In contrast to approaches that optimize Env for binding to these highly affinity-matured antibodies, we now propose to identify Env variants that can bind to the germline form of the broadly neutralizing antibodies as a more productive approach to immunization. While structural studies of antibody-antigen interactions have informed antigen design, the corresponding germline antibody sequences rarely show any binding to the HIV-1 Env. As a result, detailed information is not readily available for the rational design of germline-binding Env. We propose therefore to use a directed molecular evolution approach to identify germline-specific immunogens. Such immunogens could more effectively stimulate naive B cells. The broadly neutralizing VRC01 mAb is well suited to this approach, notably because the CDRH3 domain is less important for the neutralization activity compared to most of the other broadly neutralizing mAbs. This minimizes one of the constraints in predicting the structure of the germline VRC01 precursor. We will create a number of VRC01-like mAbs based on macaque germline sequences that are increasing reverted to the germline sequence and use these as reagents to screen libraries of Env variants created by in vitro homologous DNA recombination. We will immunize macaques with these germline-specific variants and analyze, using massively parallel DNA sequencing technologies, the ability of these Env-based immunogens to stimulate affinity maturation of the specific germline for which they were selected. Based on the results of an initial immunization study, we will identify additional immunogens that stimulate partially affinity-matured antibodies along a particular path to form a mature neutralizing antibody. The germline-specific and the additional immunogens will be used to immunize macaques over a 10-month period. Analysis of the pattern of mutation in the antibody genes will indicate whether it is possible to direct antibody maturation along a specific pathway. Even if we do not succeed in eliciting VRC01-like activity, the results of this study will lead to a greater understanding of the initial response tothe immunogen and to what extent can it be manipulated and controlled. This proposal represents an innovative approach to the identification of candidate immunogens for HIV-1 vaccines. It has general applicability to other pathogens for which protective (or broadly protective) mAbs have been identified (e.g. RSV or influenza) but where induction of those specificities has proven problematic.